The present invention is directed to fusion proteins having high affinity coiled-coil heterodimerization domains in place of a modular protein binding domain and the use in an assay system for (1) validating that a protein-protein interaction causes a specific biological activity, (2) identifying target molecules capable of affecting those interactions and (3) identifying the biological activities involved in such interactions. In particular, the present invention is directed to fusion proteins containing an exogenously introduced zipper.
Many critical cell processes are regulated by specific protein-protein interactions. These interactions can cause signal transduction or processes governing whether a cell will proliferate, differentiate, die, adhere, migrate or otherwise respond to its environment. Protein-protein interactions may entail, for example, between a receptor and its target ligand (such as VEGF165 Receptorxe2x80x94Neuropilin-1 complexes) as well as intracellular interactions (such as adaptor-kinase complexes). If one could identify in vivo binding partners, the manner in which proteins exert their activity might be discerned. If the activity of the protein is relevant to disease, then the binding proteins might also be novel targets for drug discovery. Unfortunately, in many cases a protein binds not one, but many other proteins with similar affinity. It is often therefore difficult or impossible to ascribe any functional significance to any particular interaction. This is a major stumbling block to the ability to understand the precise inner workings of the cell.
Protein-protein interactions typically involve the modular protein binding domains (MPBD) of one protein, which are regions of about 60 to 200 amino acids, and the corresponding binding site of the second protein. Examples of such domains are SH2 (src homology 2), SH3 (src Homology 3) and PTB. These domains typically bind to linear peptide epitopes of about 4-10 amino acid residues on their binding partners. MPBDs are present in a wide variety of functionally distinct proteins. For example, the SH2 domain, which binds to phosphorylated tyrosine residues, is contained in many different signaling proteins and their binding sites are found on a wide range of activated growth factors, e.g. epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, etc.
Despite the fact that numerous MPBDs and their corresponding binding sites (BSs) have been identified, these discoveries themselves do not shed light on whether a pair of proteins actually interact in vivo and whether that interaction is important for biological activity. This is because the binding interaction between MPBDs and their corresponding binding sites is not highly specific, but is only partially specific. Consequently, one protein can bind with several to hundreds of other proteins with virtually the same affinity. This lack of specificity has created a fundamental stumbling block. For example, if a single SH3 domain can bind with virtually identical affinity to tens or even hundreds of different proteins in the cell, and a single SH3 binding site can combine with tens or hundreds of different SH3 domains, which of these hundreds of potential protein complexes actually results in a specific in vivo function?
The determination of the specific pair of protein-protein interactions that results in a biologically activity is typically done indirectly, e.g., by preventing the specific interaction between the proteins to determine if preventing binding of a set of proteins eliminates a function. The latter could be performed, for example, by altering the MPBD and/or BS to prevent binding. However, such an alteration will also prevent binding with other proteins. Therefore that method does not definitively confirm that the specific biomolecular protein complex under investigation actually interacts in the cell to result in the function eliminated. For example, protein A may interact with protein 55 at one point in a pathway, whereas protein 5 interacts with protein D at a different point in the pathway. Thus, by altering the binding sites of proteins A and 5, the function could be lost without the two proteins having to interact directly with each other.
There also exists a method for studying protein-protein interactions wherein proteins are attached to one another through bifunctional molecules. In this approach, proteins are synthetised as fusions which bind a small-molecule drug, and the small-molecule drug is added. This method works well for some applications, but the protein fusion partners tend to be quite bulky and the affinities of interaction somewhat low. The dimerization drugs may also only be available by licensing from companies.
The predominant system used today for studying protein-protein interactions is the two-hybrid assay system. In this method a selectable output such as growth on a selected media, or metabolism of colorimetric substrates is dependent on reconstituting a protein-protein interaction with a xe2x80x9cbaitxe2x80x9d protein. Such a system is limited, in that the selectable biological output is fixed by the experimental system and proteins are screened for their ability to bind to the target (bait) protein. Further, one is typically trying to reconstitute function in a foreign system, e.g., yeast, as opposed to a mammalian cell system. It would be desirable to have a system that forces interactions between the proteins and looks at their functional consequences. It would also be desirable to have a system that more closely resembles the actual cellular microenvironment where the protein-protein interactions occurs.
In co-pending PCT Application No. PCT/US00/17929, filed Jun. 29, 2000 and entitled xe2x80x9cFusion Protein and Uses Thereof,xe2x80x9d the present inventor discloses a Functional Interaction Trap(xe2x80x9cFITxe2x80x9d) system which depends on a protein binding interface consisting of two engineered protein segments. One fusion protein comprises a protein containing a modular protein binding domain (MPBD), wherein the MPBD is substituted by a single chain antibody. The MPBD may be selected from the group of domains consisting of src homology 2 (SH2), src homology 3 (SH3) phosphotyrosine binding (PTB), WW, PDZ, 14.3.3, WD40, EH, Lim, etc. The second fusion protein comprises a protein containing a binding site that binds to a modular protein binding domain MPBD, wherein at least one linear epitope that binds to the MPBD within the binding site is substituted by an antigenic epitope of 6-20 amino acids that binds to the single chain antibody that has been substituted for the MPBD. Preferably, the second fusion protein contains multiple copies of the epitope. For example, 2-20, more preferably 3-15, still more preferably 4-10 copies of the epitope. Nucleic acid sequences encoding these fusion proteins can be prepared by known techniques. Preferably these sequences (e.g., genes) are contained in vectors and are operably linked to a promoter. These vectors can be used to transform a cell. When the protein fused to the ScFv interacts with the second protein fused to the epitope the two proteins bind to each other. Such interaction can only occur if both modified proteins are expressed in the same cell. The unique biological consequences of the interaction can be assessed to provide information previously unaccessible by prior art techniques.
The antibody FIT technique is not always optimal in that the specificity of the reaction between the particular ScFv and epitope may permit in the reducing environment of the cytosol reactivity with other unintended proteins. The intracellular environment does not favor the disulfide bonds that normally stabilize the structure of the antibody in the extracellular environment. In the intracellular environment ScFvs may be susceptible to unfolding and thus may be unstable and insoluble. It would be desirable to have a protein that can still perform its native functions, but can bind with even higher affinity to its putative partner than presently available. It would also be useful to have a method to replace the relatively nonspecific interactions between two proteins with a highly specific interaction, thereby allowing the two proteins of interest to directly interact without concern for competing interactions with other proteins in the cell.
It has been discovered that novel fusion proteins having high affinity coiled-coil heterodimerization domains (Kd of heterodimerization interaction less than, or equal to, 30 nM, more preferably less than, or equal to, 28 nM, and more preferably less than, or equal to, 26 nM) in place of a modular protein binding domain can be used in assays to identify the effect of protein-protein interactions.
Particularly preferred high affinity coiled-coil heterodimerization domains of the present invention are the WIN-ZIP coiled-coil heterodimerization leucine zippers, such as those described by Michnik and colleagues (See, e.g., Arndt, K. M., J. N. Pelletier, K. M. Muller, T. Alber, S. W. Michnick and A. Pluckthun, A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble, J Mol. Biol. 295:627-630 (2000).
One fusion protein of the present invention comprises a protein containing a modular protein binding domain (MPBD), wherein the MPBD is substituted by a WIN -ZIP A1 segment. Preferably, the MPBD is selected from the group of domains consisting of src homology 2 (SH2), src homology 3 (SH3) phosphotyrosine binding (PTB), WW, PDZ, 14.3.3, WD40, EH, Lim, etc. For example, such a protein may be a tyrosine kinase. A second fusion protein comprises a protein containing a binding site that binds to a modular protein binding domain MPBD, wherein at least one linear epitope that binds to the MPBD within the binding site is substituted by a WIN-ZIP B1 segment.
It is believed when two proteins are expressed in transiently transfected mammalian cells that if one is a WIN-ZIP A1 fusion, and the other WIN-ZIP B1 fusion, the two proteins will specifically bind each other and stably co-precipitate from cell lysates in a ratio close to 1:1 by forming stable heterodimerization coiled-coil structures. The present inventor has found that when the WIN-ZIP A1 fusion is the Src tyrosine kinase, a co-expressed WIN-ZIP B1 fusion is specifically phosphorylated on typrosine by Src (but is not phosphorylated to nearly the same extent if either of the two proteins does not bear the WIN-ZIP segment). Thus this system, may be used to induce the highly specific phosphorylation of particular substrates by kinases of interest, at least in the cases where the kinase is known to require substrate interaction domains to efficiently phosphorylate its substrates (this is the case for all non-receptor tyrosine kinases, cyclin-dependent kinases, MAP kinases, etc.). No other system currently available allows the assay in vivo of the consequences of phosphorylation of a single substrate of interest.
As would be understood by one of ordinary skill in the art, the FIT WIN-ZIP approach of the present invention permits the specific binding of two proteins that normally would not interact in the cell, as well as those proteins that would normally interact. It can also be used to direct an enzyme to act specifically on a single substrate protein of choice. The availability of this interface permits one to easily test the functional consequences of binding for specific pairs of proteins, and to screen large libraries of proteins for those whose interaction with a protein of choice gives some biological activity of interest.
Nucleic acid sequences encoding these fusion proteins can be prepared by known techniques. Preferably these sequences (e.g., genes) are contained in vectors and are operably linked to a promoter. These vectors can be used to transform a cell. These transformed cells can be used to identify the function of a protein-protein interaction, to identify a particular protein involved in an interaction and to study the specific effect of specific functional domains.
In one embodiment there is an assay for determining the activity of a protein-protein interaction, comprising:
(a) transforming a cell with a first vector containing a gene encoding a first fusion protein comprising MPBD and an exogenously introduced coiled-coil dimerization domain, and with a second vector, wherein said second vector contains a gene encoding a second fusion protein comprising a binding site for the MPBD and an exogenously introduced second coiled-coil dimerization domain interactive with the first coiled-coil dimerization domain;
(b) culturing the transformed cell;
(c) and comparing the activity to a base line control; and
(d) measuring changes in activity to determine the activity caused by that protein-protein interaction.
Preferably, the cell used does not express the protein containing the MPBD of the fusion protein encoded by the gene contained in the transforming vector or the effect of the interaction is dominant or assayed in a way that does not depend on the lack of the wild-type counterpart of the engineered gene. The control can be an untransformed cell, or more preferably cells transformed with each of the modified fusion proteins, alone, but not together. By this latter way, one can determine the effect of expression of each of the proteins on the cell and determine what effects are dependent on the interaction of the two proteins.
Full-length cDNAs fused to sequences encoding coiled-coil heterodimerization segments may be used in FIT screens. The coiled-coil heterodimerization segments such as WIN-ZIP are short enough that it is possible to synthesize them in vitro and couple them to various chemical groups (fluorophores, reactive groups, inhibitors, membrane-transit domains, etc.) for in vitro biochemical studies on purified enzymes or studies in tissue culture cells.